The actual lytic mechanism at the conclusion of human lymphocyte-mediated natural cytotoxicity has not been described, but it is usually assumed that a mediator is transferred from the effector cell to the target cell. We have recently made three observations that may provide insight into this event: (1)\human natural cytotoxicity can be inhibited by mannose-6-phosphate, which is a known recognition marker for some human lysosomal enzymes; (2)\there is a receptor for mannose-6-phosphate on the surface of some target cells of natural cytotoxicity; and (3)\amine inhibitors of lysosomal receptor recycling cause inhibition of natural cytotoxicity if either of the two elements (effector cell or target cell) are pretreated with these agents. A working hypothesis has been formulated that suggests that, subsequent to effector cell-target cell contact and programming for lysis, a glycoprotein mediator is expressed from the effector cell to the outside where it is bound to and internalized by the target cell by virtue of a mannose-6-phosphate recognition marker. The internalized mediator may be passed through the target cell lysosome, and it is released into the cytoplasm in a toxic form. It is intended to test this hypothesis and to extend this observation. The temporal relationship of this carbohydrate inhibition to the events of natural cytotoxicity will be explored in detail. Murine cytotoxicity against the YAC1 target cell line and its two sublines YAC 19 (NK sensitive) and YAC 28 (NK resistant) will be utilized in studies to isolate soluble mediator of cytotoxicity. YAC 28 has been shown to form conjugates with NK cells but appears to be resistant to the lytic consequences. YAC 28 is also relatively resistant to a conjugate of gelonin (a plant toxin with no means of entry into the cell) and pentamannose-6-phosphate (a mannan from Hansenula holstii), which suggests that this cell line is deficient in internalizable surface receptors for Man-6-P. It is intended to use this target cell line as a model in which the target cell can potentially bind to the effector cell and initiate programming for lysis but in which the target cells are also unable to bind the lytic moiety that is subsequently secreted from the effector cell. This provides an ideal environment in which to isolate and characterize partially this lytic moiety. (LB)